Thrombotic Thrombocytopenia Purpura (TTP) is a life threatening thrombotic microangiopathic disease characterized by hemolytic anemia and thrombocytopenia associated with platelet aggregation. The cause of TTP has been recently linked to abnormalities in a metalloproteinase called ADAMTS13 or von Willebrand factor cleaving protease. ADAMTS13 is an enzyme that is present in significant levels in plasma, and may be expressed in other tissues (Levy et al. 2001, Nature 413:488-494; Plaimauer et al. 2002, Blood 100:3626-3632). Suzuki et al. (2004, Biochem. Biophys. Res. Com. 313:212-216) recently reported ADAMTS13 in platelets.
ADAMTS13 functions by cleaving ultralarge von Willebrand factor (VWF) multimers to smaller VWF proteins. Decreased VWF cleaving protease activity leads to persistence of unusually large multimers of VWF that bind to platelets, causing platelet aggregates, microangiopathic hemolysis, and thrombocytopenia in patients with TTP. Clinical manifestations of TTP are difficult to distinguish from hemolytic uremic syndrome (HUS), another thrombotic microangiopathic disorder. Recent studies indicate that low levels of ADAMTS13 activity are associated with TTP, but not HUS (Veyradier A, et al. 2002, Blood 98:1765-1772; Furlan et al. 1998, Blood 91:2839-2846). Thus, differential diagnosis of TTP can be made by measuring ADAMTS13 activity.
There are two forms of TTP—congenital (familial) and acquired (Furlan et al. 1996, Blood 87:4223-4234; Furlan et al. 1998, Blood 91:2839-2846). Congenital TTP is caused by genetic mutations in the ADAMTS13 gene, which result in a loss in ADAMTS13 production and/or the production of a non-functional ADAMTS13 enzyme. Acquired TTP is an autoimmune-like disease, which has been linked to intake of certain pharmaceutical drugs. Acquired TTP is caused by the generation of autoantibodies to ADAMTS13 protein. The onset of acquired TTP has been linked to intake of certain pharmaceutical drugs.
Several methods for measuring the presence and/or activity of ADAMTS13 are known in the art. These methods include collagen binding assays, ristocetin cofactor assays, electrophoretic analysis (e.g. multimer analysis) and immunological methods. Electrophoretic immunoassays, such as Western blotting analysis, have largely been replaced by immunoradiometric assays (IRMA) and enzyme-linked immunosorbant assays (ELISA) (Laffan et al. 2004, Haemophilia 10:199-217). Several reports describe ADAMTS13 assays where the A2 domain of VWF is used as a substrate (Zhou et al. 2004, Thromb. Haemost. 91:806-811; Kokame et al. 2004, Hemost. Thromb. Vasc. Biol. Blood 103:607-612; Cruz et al. 2004, Thromb. Haemost. 90:1204-1209). Cleavage of the A2 domain is monitored by an ELISA method. The available assays for ADAMTS13 require a relatively high technical skill level, and are therefore not performed in most clinical laboratories. In addition, obtaining results can take several days using available tests, which is detrimental to patients presenting with TTP, who require rapid diagnosis.
A simple, specific and rapid assay for ADMTS13 is needed to solve the problems with the currently available assays; yet one has not been developed to date. Attempts at developing such an assay using ADAMTS13 isolated from plasma have not been successful. Furlan et al. tested 28 synthetic chromogenic peptidyl substrates with para-nitroanaline (pNA) as the leaving group, and did not observe consistent, repeatable results (2002 Seminars in Thrombosis and Hemostasis 28(2):167-172). These results demonstrate the difficulty in the art of developing a rapid, reliable assay for ADAMTS13 activity.
We have discovered that ADAMTS13 on platelets has an enhanced ability, relative to ADAMTS13 in plasma, to cleave small peptidyl substrates. The difference between plasma and platelet ADAMTS13 is likely due to the finding that platelet ADAMTS13 is cleaved, while plasma ADAMTS13 is not cleaved and remains a single polypeptide. The ADAMTS13 activity on platelets can be enhanced by treatment with coagulation Factor XIa (FXIa). This indicates that activated FXI (FXIa) may cause cleavage of ADAMTS13. We have developed diagnostic chromogenic assays for measuring ADAMTS13 activity on platelets and in plasma using different peptidyl substrates with leaving groups other than pNA. We have also developed an assay method to measure autoantibodies to ADAMTS13 and an ELISA for measuring ADAMTS13 protein in plasma.